The Marfan syndrome is an autosomal dominant disorder of connective tissue with an estimate prevalence of 1/10,000 people. Dilatation, dissection and rupture of the proximal aorta are the most serious complications of the disorder, other phenotypic features, include ectopia lentis, scoliosis, dural ectasia, and spontaneous pneumothorax. The cause of Marfan syndrome is uncertain, and the molecular basis for these extensive pleiotropic manifestations remains unknown. In the past year, several laboratories have presented immunohistochemical and biochemical evidence pointing to fibrillin, a 350 kD protein found in the microfibrils, as the leading candidate for the mutant molecule in the Marfan syndrome. In the same year, we showed that the Marfan gene is tightly linked to D15S1, an anonymous DNA fragment mapped to human chromosome 15ql5-2l.3. The overall goal of this proposal is the identification of the gene causing the Marfan syndrome and the characterization of mutations in this disorder. The specific aims of this proposal are to (1) develop a long-range restriction map around the Marfan syndrome locus, as best defined currently by D15SI, (2) determine the physical relationship between the Marfan locus, D15SI and the fibrillin gene, (3) assess whether mutations at the fibrillin locus on chromosome 15 cause the Marfan syndrome (4) if the fibrillin locus is not the Marfan locus, identify and characterize the Marfan syndrome locus, and (5) identify and characterize mutations in patients with the Marfan syndrome. Restriction fragments resolved using both pulsed-field gel electrophoresis and "conventional" Southern blot techniques will be analyzed to accomplish specific aims 1 and 2. The physical relationships between D15SI and fibrillin on pulsed-field maps should determine whether the loci are distinct. Mutations will be detected by hybridization of D15S1 and fibrillin cDNA clones to pulsed-field gel blots and Southern blots to look for gross alterations in gene structure. Fibrillin cDNA synthesized from mRNA isolated from Marfan fibroblasts will be amplified by PCR and searched for evidence of mutation using denaturing gradient gel electrophoresis (DGGE) or non-denaturing strand separation techniques. Amplified fragments exhibiting evidence of mutation will be sequenced. If, as expected, substantial genetic heterogeneity is discovered, genotype-phenotype relationships will be explored as one explanation of interfamilial variability of the disorder. Identification of the genetic locus causing the Marfan syndrome and identification and characterization of Marfan mutations are likely to have important clinical implications (e.g. diagnosing presymptomatic persons at risk and isolated cases who may or may not have the full syndrome with its attendant risks), as well as enhancing understanding of the development and pathobiology of the diverse tissues involved in the disorder.